Webinar One High Throughput Quantitative Analysis · In analytical chemistry, quantitative analysis...
Transcript of Webinar One High Throughput Quantitative Analysis · In analytical chemistry, quantitative analysis...
High Throughput Quantitative Analysis
Howard Sanford
Stephen HarnosWith Tom Barrett & Matt Leyden & Kevin Costalunga
MassHunter Quantitative Analysis Webinar Series
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What is quantitative analysis?
In analytical chemistry, quantitative analysis is the determination of the absolute or relative abundance (often expressed as a concentration) of one, several or all particular substance(s) present in a sample.
From https://en.wikipedia.org/wiki/Quantitative_analysis_(chemistry)
Quantitative chemical analysis, branch of chemistry that deals with the determination of the amount or percentage of one or more constituents of a sample.
From https://www.britannica.com/science/quantitative-chemical-analysis
Quantitative analysis refers to the determination of how much of a given component is present in a sample.
From https://www.thoughtco.com/definition-of-quantitative-analysis-604627
How do I know that the compound that is identified is indeed the correct compound?’
MassHunter Quantitative Analysis SoftwareReview and Quant Method Optimization
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• A given compound will come out at a specified time under a given set of chromatographic conditions.
• Dual column analysis with columns of different polarities.Retention Time
• Must be unique to the compound of interest in the time range of interest.Target Ion
• Must be present in the same specified time range.
• Must be present in specific ratios relative to the Target Ion.
Qualifier Ions
• Must be within the given correlation window.
• Should maintain similar peak shapes.Target and Qualifiers
Target Compound Analysis
Increasing Confidence
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Analysts are buried in data!!!
Analysts are being overwhelmed with data … hundreds of compounds… multiple signals per compound…10’s or even hundreds of samples in a batch.
Add an initial calibration, a continuing calibration or a QC and other data review functions and the analyst is overwhelmed with data.
Chromatographic runs are shorter; thus more data in a given period of time.
What can what can MassHunter Quantitative
Analysis software do to help?
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Quant Method Check List
• Extract the compounds signals in the correct RT Window.
• Utilize the correct integrator and settings for each compound.
• Reduce unwanted peaks with
• Reference and Non Reference Windows
• Integration parameters
– Peak Filters
– Peak Filter Area Threshold
– Zero Peaks Below LOD
– Correlation Window
• Updating Retention Time Drift
• Retention Times
• Qualifier Ion Ratios
• Update Mass Assignments
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Objective is to intelligently minimize the number of compounds that require review.
Retention Time SetupRT Delta
Left and Right RT Delta
determines the time range over
which the specified signal is
extracted.
RT Delta Units• Percent – Uses a percentage of RT,
good for long runs where retention
time shifts can be larger late in the
run.
• Minutes - Absolute minutes.
Default is 1 minute. A narrower
window can be setup if needed.
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Retention Time SetupCriteria
When multiple peaks are found
in RT window, Criteria decides
which peak to use.
• Close RT
• Close RT with Qualifiers
• Greatest Response
• Greatest Q-Value
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Reference & Non Reference WindowDefinitions
Defined in Globals Setup in the
Method Editor
Reference Window
• Applies only to compounds labeled as
Time Reference.
• Only ISTDs can be labeled as Time
Reference.
• Algorithm looks for ISTDs first, then
target compounds related to that
ISTD.
Non Reference Window
• Applies to all other compounds.
Recognition and Reference Windows are
synonymous terms.
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Reference & Non Reference Window
Restricts peak selection to a smaller RT window.
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1
2
3
Non Reference Window
Reduce the Non Reference Window from the default 200% to 10%. This helps eliminate false
positives and reduced data review and “zero peak” work.
Or, if you prefer, switch to absolute minutes. Though keep in mind this setting is for all peaks
in the quantitation method and cannot be changed on a compound by compound basis.
Initial
ValuesFinal
Values
Adjusted
Values
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Properties Window
Right click Properties > Fill Colors.
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Integrators Choose the Right Integrator
• Method >Edit >Advanced Tasks >
Integration Parameters Setup
• Each compound can have its own integrator.
• Choose the one best suited for the compound’s
chromatography.
• Start with Agile2 (parameter-less) and move to
other integrators if they work better.
• All integrators use Peak Filters.
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IntegratorsAgile2
• 3rd generation parameter-less integrator
• Default Integrator
• Better baselines, higher sensitivity to smaller peaks
Agile
• 2nd generation parameter-less integrator
Universal
• 1st generation ChemStation integrator
• Familiar to GC LC ChemStation users
General (RTE)
• Familiar to MSD ChemStation users
• Areas in Universal are 10 time smaller than seen in ChemStation.
MS/MS and MS/MS (GC)
• 1st generation parameter-less integrator intended for MS/MS systems, not recommended for SQ. Originally required 64 data points.
ChemStation
• 2nd generation ChemStation
• Intended for UV
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Integrators General, Universal, Spectrum Summation
Adjust parameters as
needed for each compound
to get optimal integrations.
Timed Events
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Integrators Qualifier Integration Control
Qualifiers use the same
integrator as quantifier.
If needed each qualifier may
have separate parameters to
optimize integration but the
default is to use quantifier
parameters.
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Spectrum Summation Integrator
• Integrator designed for situations where compounds are poorly separated or peak shape is highly irregular such as ….
– PCB mixtures
– Fraction cut in hydrocarbons
– Flow injection analysis (FIA)
• Sums signal over a time range.
• Exclude signal below threshold.
• Always gives a horizontal baseline.
• RT reported as the center of the time range.
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Integrators Peak Filters
• Available on all integrators including
parameter-less ones.
• Separate Peak Filters for quantifier and
qualifiers.
• Peak Filter is run after integrators create a
peak list and then removes peak based on
settings.
• Ideal to automatically remove small peaks
that would otherwise require manual review
and correction.
Default Setting for both Target and
Qualifiers is Peak Area >= 5 % of
largest peak.
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Peak FilterArea
Typical noise or matrix peaks may be
picked up by the integrator.
They are far too small relative to the
response of the lowest level Calibrator
to be reported. Normally we would
need to “zero peak” each one.
Instead we can use peak filters in the
method to remove the unwanted peaks.
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Adjusting Peak Filter Area ThresholdsManual
Setting the peak area threshold
for each compound would be a
slow and tedious process…
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Adjusting Peak Filter Area ThresholdsAutomated
To automate the process, while in the Method Editor
view, use the “Set Peak Filter Area Threshold” script to
set each compound to a percent of its lowest Calibrator
in the batch.
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Adjusting Peak Filter Area ThresholdsExample
Typical noise or matrix peaks are removed and require no data review or “zero peak” work.
Before After
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Zero Peak Below LOD
Method Editor > Outlier Setup Tasks >
Limit of Detection.
• Eliminate peaks based on concentration
rather than area.
• Must define the Limit of Detection outlier value for each compound.
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Zero Peak Below LOD
Tools > Actions > Zero Peak Below LOD
• Accessed from Batch Table View or Method
Editor View.
• Zeros compounds if the Calculated
Concentration is less than the LOD.
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Correlation WindowThe retention time difference limit of target ions to one or more qualifiers.
• Defines the maximum allowable variation of multiple extracted ion peak retention times before they are considered a single peak.
• Default time of 2.00 min is rather wide. Typically 0.01 to 0.05 min (0.6 sec to 3.0 sec).
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Correlation Window
Note that the retention times for target and qualifiers are different!
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Default value = 2.0
minutes.
Retention Time Drift
Retention times will Drift
• GC
‒ Can use RTL locking to mitigate drift.
‒ Use time reference compound in the middle of chromatographic run.
‒ Make a single compound standard in the mid range of concentration.
‒ Lock the method.
‒ Relock the method as necessary.
• LC
‒ Utilize a guard column.
‒ Sample preparation steps.
‒ Change solvents frequently to avoid microbial growth.
‒ Clean solvent bottles when changing solvent.
But…retention times will still drift.
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Retention Time DriftUpdate Retention Times
Update Retention Times is available in Batch Table View and Method Editor View.
Caveat - the compound must be found to update it.
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Retention Time DriftUpdate Retention Times from ISTD
• Update Retention Times from ISTD is a Method Editor feature.
• Particularly useful with isotopically labeled ISTD.
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Retention Time DriftAverage Retention Times
• Average Retention Times is a Method Editor feature.
• Allows choice of Cals or QCs or both.
• Includes a weighted average.
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Retention Time DriftShift Retention Time
• Shift Retention Time is a Method Editor feature.
• Allows an Absolute Shift in minutes or a Relative Shift in percentage.
• Would be most applicable when changing columns.
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Qualifier Ion Ratios
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Update Qualifier Ratios
Or
Average Qualifier Ratios
Can use Cals and/or QCs.
Based on the sample currently loaded in Method Editor.
Update Mass Assignments
• Update Mass Assignments is a Method Editor feature.
• It is based on currently selected sample in Batch Table.
• Useful with high resolution data (TOF and QTOF).
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Reference LibraryGlobals Settings Option
• Activated from Method > Edit > Library > Setup Reference Library
• Reference Library may be obtained from a sample, preferably a calibrator, or from a small user
generated library or a small subset library.
• Reference Library name is auto populated in Globals Settings.
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Reference LibraryGlobals Settings Option
Appears in Compound Information window.
Customizable in right click > Properties > Compound Information (2) tab.
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Reference Pattern LibraryHigh Resolution Data
• Activated from Method > Edit > Library > Setup Reference Pattern Library.
• Can be obtained from a calibrator or from another library.
• Method must contain molecular formula.
• Reference Pattern Library name is populated in Globals.
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Select the adducts.
Reference Pattern LibraryHigh Resolution Data
• Right click Properties > Compound Information (2) > Reference pattern spectrum
• Isotopic abundance and pattern appears in Spectrum window.
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Demo time
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Data Review Check List
Filter on Sample Type.
Filter on Sample Group.
Filter on Compound
Group.Auto Review.
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Data ReviewFilter on Sample Type
Review Data in stages (Calibrators, then QC, then Samples).
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Focus on only the calibrators
or samples or blanks, etc.
Data ReviewSample Type
• The Type or Sample Type is a parameter available in the sequence or worklist.
• Should be specified when data is acquired, but if necessary it can be entered in the Batch Table.
• In Sequence Table in GCMS software.
• In Worklist in LC MassHunter software.
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Data ReviewFilter on Sample Group
• This is helpful with large batches that contain several sample types.
• Sample Group is a column that can be added to the Worklist or Sequence Table.
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Sample Group is activated by
right clicking on the toolbar.
Data ReviewFilter on Compound Group
• Compound groups are useful for parent compound and metabolites.
• Reviewing Aroclor congeners by group (PCB).
Compound groups are assigned in the Method Editor.
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Data ReviewFilter on Compound Group
Review Compounds by Group (using Compound Table View).
Compounds may be assigned to more than one group by separating the group
names using commas.
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Data ReviewCompound Information
Manual Integration turned on.
Compound groups are shown in the Compound information window.
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BHC Isomers (3)
Data ReviewChromatogram Information
• Accessed from View > Chromatogram Information.
• Useful to compare multiple chromatograms.
• Useful to compare patterns.
• Create Compounds GC Data only Method Editor.
• Available for GC and MS Quantitative Analysis only.
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Data ReviewChromatogram Information
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Head to head view.
Can anchor a chromatogram.
Data ReviewChromatogram Information
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Head to tail view.
Colors are changeable.
Data ReviewChromatogram Information
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Overlay view.
Many other options are
available.
Auto Review
Auto Review Samples
• Displays sample by sample.
Auto Review Compounds
• Displays compound by compound.
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• Available in Flat Table or Compound
Table.
• Stop, pause, continue and variable
intervals.
Demo time
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Outlier Check List
An outlier is a result that is outside the range of acceptable values for a given parameter.
What outliers are important in the workflow?
• Retention Time…
• Limit of Detection, Quantitation, Method Detection Limit.
• Qualifier Ratio.
• ISTD Response or ISTD Response Percent Deviation.
• QC, QC Relative Standard Deviation, QC LCS Recovery…
• CC, CC Average Response Factor, CC ISTD Response Ration…
• Matrix Spike, Matrix Spike Percent Recovery….
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Outliers Setup Tasks
Outliers are setup in the
Method Editor and are part
of quantitation method.
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Method > Edit >Outlier
Setup Tasks
Outliers Setup Tasks
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~ 48 Outliers are available.
Outliers are not calculated unless values have been set up.
Outliers are used to perform automated quality checks.
Aids in data review by highlighting problem areas.
Increases confidence in data integrity by utilizing outliers.
Which outliers are important for my workflow?
Outliers
• Most Outliers have both a low and high limit.
• Both limits need to be set
for the outlier to be
calculated.
• Some outliers are one dimensional.
• A few only have a single limit.
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OutliersBatch Table
Icons on the toolbar.
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Select Outliers
Turn off outlier filter
Display rows that have High/Low
outliers
Display rows that have High outliers
Display rows that have Low outliers
Display rows that have no outliers
OutliersMessages and Outliers
Messages
Outliers
Red Outlier – High
(above upper limit)
Select
Outliers
for Display
Blue Outlier – Low
(below lower limit)
Hover cursor over the outlier or message to display details
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OutliersFilter on Outliers in Batch Table
By default all outliers are displayed but it is possible to limit this to a subset or single outlier for a more targeted data review pass.
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Remember, an outlier is not active unless a limit has been specified for it in the Method Editor.
OutliersDefault outliers
By default these outliers are enabled.
If an outlier is not enabled, it is not calculated.
Accuracy = Calculated/Expected x 100
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OutlierHelp
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Many Outliers also have Quant videos.
Metric Plot
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Right click (on header) > Plot this Column.
Right click (in the plot window) >
Show Average/Std Dev lines.
Compounds at a Glance
Custom layouts can
be loaded and saved.
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View > Compounds-at-a-Glance
Compounds at a Glance
Layout > Setup Layout
Select various Qualifiers
Show panes with or without outliers
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Display outliers by category.
Compounds at a GlanceData Review
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Sample Approved
box for QA.
Compounds at a GlanceReview Sample by Sample
• Can be reviewed Sample by Sample.
• Optional Pane Dimensions
• Can scroll through the samples.
• Once the layout is saved it can be loaded time after time.
• Numerous Predefined Layouts
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Compounds at a GlanceReview Compound by Compound
• Layout > Setup Layout or from
• Layouts can be customized.
• Can be reviewed Compound by Compound.
• Various Overlay modes.
• Various Display Options.
• Can synchronize Compounds at a Glance with Quantitative Analysis.
• Right click Properties > Synchronize Navigation (global parameter).
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Demo time
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Summary
• Target Compound Analysis
• Quant Method Checklist
• RT setup and RT Criteria
• Reference and Non-Reference windows
• Integrators
• Peak Filters
• Adjusting Peak Filter Area Thresholds Zero Peak Below LOD
• Correlation window
• Data Review
• Filtering on Sample Type, Sample Group and Auto Review
• Outliers
• ~ 48 outliers are available
• Which outliers are important to my workflow?
• Compounds at a Glance
Display outliers by category.
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